A protocol for isolating pure DNA from goat ruminal fluid was developed. DNA was partially digested using ApoI enzymes and ligated onto Lambda ZAP II Predigested EcoR l/CIAP-treated vector to make a metagenomic DNA library. DNA sequence analyses showed that positive clones carried complete or partial sequences for structural genes in different strains of Escherichia coli. Several clones did not share significant similarity with nucleotide in the databases. Research results indicate that the population of prokaryotes in the goat rumen contained E. coli, and possibly other organisms that have not been characterized. This is the first report of the use of molecular approaches to characterize microbial genes or organisms from goat rumens.